Assays and methods for detecting udp-glucose

Abstract

Disclosed herein are methods, assays, compositions and kits for the detection and measurement of UDP-glucose in biological samples. The methods, assays, compositions and kits are suitable for use with equipment that is readily available in a clinical laboratory, and permit rapid and reproducible detection and measurement of UDP-glucose at physiologically relevant levels in biological samples.

1 . (canceled) 
     
     
         2 . A composition comprising uridine diphosphate glucose (UDP-glucose) dehydrogenase immobilized on a solid support. 
     
     
         3 . The composition of  claim 2 , wherein the solid support is selected from the group consisting of a cell culture plate, a multiwell plate, a disc, a bead, a woven paper, a filter paper, cardboard, a well, a plate, an electrode, a coated test strip, an uncoated test strip, a lateral flow strip, a lateral flow device, a dipstick device, a particle, and a magnetic particle. 
     
     
         4 . The composition of  claim 3 , wherein the solid support is a lateral flow strip, lateral flow device, or dipstick device. 
     
     
         5 . The composition of  claim 4 , wherein the lateral flow strip, lateral flow device, or dipstick device comprises a test region buffered to pH 8-9. 
     
     
         6 . The composition of  claim 5 , wherein the test region comprises NAD+. 
     
     
         7 . The composition of  claim 5 , wherein the test region comprises immobilized UDP-glucose dehydrogenase. 
     
     
         8 . The composition of  claim 5 , wherein the lateral flow strip, lateral flow device, or dipstick device comprises a region with a strong base or a region of immobilized NAD+ nucleosidase. 
     
     
         9 . The composition of  claim 8 , wherein the lateral flow strip, lateral flow device, or dipstick device comprises a region with a strong base. 
     
     
         10 . The composition of  claim 9 , wherein the region with a strong base comprises a high pH membrane. 
     
     
         11 . The composition of  claim 8 , wherein the region of strong base or the region of immobilized NAD+ nucleosidase is positioned following the test region in the direction of capillary flow in the lateral flow strip, lateral flow device, or dipstick device. 
     
     
         12 . The composition of  claim 8 , wherein the lateral flow strip, lateral flow device, or dipstick device comprises a detection reagent that provides an optically detectable readout product in the presence of NADH. 
     
     
         13 . The composition of  claim 12 , wherein the detection reagent is nitro blue tetrazolium (NBT), a luciferin reagent, or a fluorescence detection agent. 
     
     
         14 . The composition of  claim 12 , wherein the detection reagent is positioned following the test region and following the region of strong base or the region of immobilized NAD+ nucleosidase in the direction of capillary flow. 
     
     
         15 . The composition of  claim 12 , wherein the detection reagent is positioned directly after the test region in the direction of capillary flow. 
     
     
         16 . A composition comprising uridine diphosphate glucose (UDP-glucose) dehydrogenase immobilized on a solid support selected from the group consisting of a lateral flow strip, lateral flow device, or a dipstick device, wherein the lateral flow strip, lateral flow device, or dipstick device comprises the following regions in order:
 (1) a sample application zone where the sample is applied and moving in the direction of capillary flow;   (2) a test region comprising immobilized UDP-glucose dehydrogenase;   (3) a region with a strong base or a region comprising immobilized NAD+ nucleosidase; and   (4) a region comprising a detection reagent that provides an optically detectable readout product in the presence of NADH.   
     
     
         17 . The composition of  claim 16 , wherein the test region is buffered to pH 8-9. 
     
     
         18 . The composition of  claim 16 , wherein the test region comprises NAD+. 
     
     
         19 . The composition of  claim 16 , wherein the lateral flow strip, lateral flow device, or dipstick device comprises a region with a strong base. 
     
     
         20 . The composition of  claim 19 , wherein the region with a strong base comprises a high pH membrane. 
     
     
         21 . The composition of  claim 15 , wherein the detection reagent is nitro blue tetrazolium (NBT), a luciferin reagent, or a fluorescence detection agent.